Following infection of a permissive cell, influenza viral cytoplasmic mRNA's are transcribed from the virion RNA segments. We showed previously that both uninterrupted (colinear) and interrupted mRNA species are derived from viral RNA segments coding for the matrix protein (M) and non-structural proteins (NS). To demonstrate that interrupted mRNA's are generated by splicing, cloned cDNA was inserted into an SV40 vector so that the late SV40 transcription signals directed the synthesis of (+) strand NS RNA sequences. Poly A-containing RNA from African green monkey kidney cells infected with the SV40-NS recombinant was analyzed for the presence of NS sequences. A colinear and an interrupted NS RNA species were produced. The interrupted NS RNA contained a splice sequence at the juncture similar to that of NS2 mRNA found during influenza infection. We examined the NS polypeptides produced by the SV40-NS recombinant using immunoprecipitation and gel electrophoresis. As predicted, the NS1 and NS2 of influenza virus were detected. Specific splicing from the transcript of SV40-NS DNA ruled out the possibility that influenza mRNA's are formed by polymerase "jumping" at the consensus sequences during replication. The presence of colinear as well as spliced NS mRNA's establishes that processing occurs during in vivo influenza virus transcription.